IDEAS home Printed from https://ideas.repec.org/a/nat/natcom/v10y2019i1d10.1038_s41467-019-09005-3.html
   My bibliography  Save this article

A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae

Author

Listed:
  • Yueping Zhang

    (Beijing University of Chemical Technology)

  • Juan Wang

    (Beijing University of Chemical Technology)

  • Zibai Wang

    (Beijing University of Chemical Technology)

  • Yiming Zhang

    (Beijing University of Chemical Technology)

  • Shuobo Shi

    (Beijing University of Chemical Technology)

  • Jens Nielsen

    (Beijing University of Chemical Technology
    Chalmers University of Technology
    Technical University of Denmark)

  • Zihe Liu

    (Beijing University of Chemical Technology)

Abstract

With rapid progress in DNA synthesis and sequencing, strain engineering starts to be the rate-limiting step in synthetic biology. Here, we report a gRNA-tRNA array for CRISPR-Cas9 (GTR-CRISPR) for multiplexed engineering of Saccharomyces cerevisiae. Using reported gRNAs shown to be effective, this system enables simultaneous disruption of 8 genes with 87% efficiency. We further report an accelerated Lightning GTR-CRISPR that avoids the cloning step in Escherichia coli by directly transforming the Golden Gate reaction mix to yeast. This approach enables disruption of 6 genes in 3 days with 60% efficiency using reported gRNAs and 23% using un-optimized gRNAs. Moreover, we applied the Lightning GTR-CRISPR to simplify yeast lipid networks, resulting in a 30-fold increase in free fatty acid production in 10 days using just two-round deletions of eight previously identified genes. The GTR-CRISPR should be an invaluable addition to the toolbox of synthetic biology and automation.

Suggested Citation

  • Yueping Zhang & Juan Wang & Zibai Wang & Yiming Zhang & Shuobo Shi & Jens Nielsen & Zihe Liu, 2019. "A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae," Nature Communications, Nature, vol. 10(1), pages 1-10, December.
  • Handle: RePEc:nat:natcom:v:10:y:2019:i:1:d:10.1038_s41467-019-09005-3
    DOI: 10.1038/s41467-019-09005-3
    as

    Download full text from publisher

    File URL: https://www.nature.com/articles/s41467-019-09005-3
    File Function: Abstract
    Download Restriction: no

    File URL: https://libkey.io/10.1038/s41467-019-09005-3?utm_source=ideas
    LibKey link: if access is restricted and if your library uses this service, LibKey will redirect you to where you can use your library subscription to access this item
    ---><---

    Citations

    Citations are extracted by the CitEc Project, subscribe to its RSS feed for this item.
    as


    Cited by:

    1. Liu, Zihe & Moradi, Hamideh & Shi, Shuobo & Darvishi, Farshad, 2021. "Yeasts as microbial cell factories for sustainable production of biofuels," Renewable and Sustainable Energy Reviews, Elsevier, vol. 143(C).
    2. Hui Liu & Pei Zhou & Mengya Qi & Liang Guo & Cong Gao & Guipeng Hu & Wei Song & Jing Wu & Xiulai Chen & Jian Chen & Wei Chen & Liming Liu, 2022. "Enhancing biofuels production by engineering the actin cytoskeleton in Saccharomyces cerevisiae," Nature Communications, Nature, vol. 13(1), pages 1-14, December.
    3. Dixit, Yatika & Yadav, Preeti & Sharma, Arun Kumar & Pandey, Poornima & Kuila, Arindam, 2023. "Multiplex genome editing to construct cellulase engineered Saccharomyces cerevisiae for ethanol production from cellulosic biomass," Renewable and Sustainable Energy Reviews, Elsevier, vol. 187(C).
    4. William M. Shaw & Lucie Studená & Kyler Roy & Piotr Hapeta & Nicholas S. McCarty & Alicia E. Graham & Tom Ellis & Rodrigo Ledesma-Amaro, 2022. "Inducible expression of large gRNA arrays for multiplexed CRISPRai applications," Nature Communications, Nature, vol. 13(1), pages 1-10, December.
    5. Qun Yue & Jie Meng & Yue Qiu & Miaomiao Yin & Liwen Zhang & Weiping Zhou & Zhiqiang An & Zihe Liu & Qipeng Yuan & Wentao Sun & Chun Li & Huimin Zhao & István Molnár & Yuquan Xu & Shuobo Shi, 2023. "A polycistronic system for multiplexed and precalibrated expression of multigene pathways in fungi," Nature Communications, Nature, vol. 14(1), pages 1-10, December.

    More about this item

    Statistics

    Access and download statistics

    Corrections

    All material on this site has been provided by the respective publishers and authors. You can help correct errors and omissions. When requesting a correction, please mention this item's handle: RePEc:nat:natcom:v:10:y:2019:i:1:d:10.1038_s41467-019-09005-3. See general information about how to correct material in RePEc.

    If you have authored this item and are not yet registered with RePEc, we encourage you to do it here. This allows to link your profile to this item. It also allows you to accept potential citations to this item that we are uncertain about.

    We have no bibliographic references for this item. You can help adding them by using this form .

    If you know of missing items citing this one, you can help us creating those links by adding the relevant references in the same way as above, for each refering item. If you are a registered author of this item, you may also want to check the "citations" tab in your RePEc Author Service profile, as there may be some citations waiting for confirmation.

    For technical questions regarding this item, or to correct its authors, title, abstract, bibliographic or download information, contact: Sonal Shukla or Springer Nature Abstracting and Indexing (email available below). General contact details of provider: http://www.nature.com .

    Please note that corrections may take a couple of weeks to filter through the various RePEc services.

    IDEAS is a RePEc service. RePEc uses bibliographic data supplied by the respective publishers.